The invention relates to a nanocarrier system according to the preamble of claim 1, a compound being suited as entity carrier according to the preamble of claim 4, the uses of such a compound according to the preamble of claims 9 and 10, a kit comprising such a compound according to the preamble of claim 14 and application methods according to claims 15 and 17.
Gene therapy provides great opportunities for treating diseases like genetic disorders, infections, and cancer (T. G. Park et al. Advanced Drug Delivery Reviews, 2006, 58, 467-486). Double stranded RNA (dsRNA) induces sequence-specific post-transcriptional gene silencing by a process known as RNA interference (RNAi). The mediators of RNAi are small interference RNA (siRNA) segments of 21 to 25 base pairs in length.
These siRNAs bind to a ribonuclease complex called RNA-induced silencing complex (RISC) that guides the siRNA to its homologous mRNA targets. As a result, the bounded mRNA is cleaved; degradation of the mRNA results in gene silencing.
To achieve successful gene therapy, development of proper gene delivery systems is the main obstacle. For the uptake of DNA/siRNA various systemic and cellular barriers have to be circumvented.
A large variety of cationic compounds were reported to efficiently deliver nucleic acids or other biomolecules or even other substances such as metal ions into the cell. Generally, cationic compounds are needed to carry nucleic acids into a cell since the latter show an overall negative charge (due to their phosphate backbone) so that a charge interaction between the carrier and the nucleic acid to be carried can occur.
One of the most powerful and versatile families of carriers are polyamines. However, these polyamines exert rather high cell toxicity and low biocompatibility. Therefore, polyamines are not well suited as carriers for in vivo applications.
Cationic lipids, such as the HiPerFect reagent of Qiagen, Hilden, Germany, are also used as carrier compounds. In this context, HiPerFect is the benchmark reagent for in vitro transfections. However, due to its cell toxicity, it is not well suited as siRNA carrier for in vivo applications.
Other siRNA carriers include the RNAiFect reagent (Qiagen), DOTAP (Roche), lipofectamine (Gibco) and polyethylene imine. All compounds show also significant cell toxicity and are thus only suited for in vitro applications.
Roller et al. (S. Roller, H. Zhou, R. Haag, Molecular Diversity, 2005, 9, 305-316) describe different amine-substitued polyglycerol-based polymeric scaffolds. However, no use of these compounds as carriers has been proposed hitherto. In addition, N-Benzyl-O-polyglyceryl carbamate described by Roller et al. is not at all suited as carrier since it is too hydrophobic and is not water soluble.
Tziveleka et al. (L.-A. Tziveleka, A.-M. G. Psarra, D. Tsiourvas, C. M. Paleos, International Journal of Pharamceutics, 2008, 356, 314-324) describe five derivatives of hyperbranched polyether polyols being functonalized with quarternary or tertiary ammonium groups. These derivatives may be used to carry plasmidic DNA (pDNA).
For a transfection to be therapeutically successful, it is imperative that polymeric scaffolds to be used as carriers exert reduced cell toxicity and higher biocompatibility.
It is an objective of the invention to provide novel compounds being suited as carriers for diverse substances, also in vivo, as well as to provide a nanocarrier system and to provide a novel use of already known compounds.
This objective is attained by a nanocarrier system according to claim 1. Such a nanocarrier system comprises at least one nanocarrier being a compound having a structure according to formula (I),
wherein PG denotes a linear or branched polyglycerol core, and    X being
preferably
                and being covalently bound to a carbon atom of the polyglycerol core, wherein the polyglycerol core carries a plurality of groups of the type X,            R1 being H, linear or branched C1-C10-Alkyl, which may be substituted and/or interrupted by one or more oxygen, sulphur and/or nitrogen atoms, or R3,    R2 being H, linear or branched C1-C10-Alkyl, which may be substituted and/or interrupted by one or more oxygen, sulphur and/or nitrogen atoms, or R3,    R3 being
    R4 being H or C1-C4-Alkyl, which may be substituted and/or interrupted by one or more oxygen, sulphur and/or nitrogen atoms, and    n being 1 to 100.
The nanocarrier system further comprises at least one entity to be carried by and bound to said nanocarrier in a covalent, ionic or complexed manner, wherein said entity is chosen from the group comprising nucleotides, nucleosides, linear or circular single or double stranded oligonucleotides, oligomeric molecules comprising at least one nucleoside, small pharmacologically active molecules having a molecular mass of not more than 800 g/mol, amino acids, peptides, and metal ions.
In this context, the entity of the claimed nanocarrier system does preferably not comprise double stranded circular and covalently closed nucleic acid species (preferably DNA or RNA, in particular DNA) having a length of more than 1000 bases or base pairs, preferably of more than 500 bases or base pairs. Thus, plasmidic DNA, i.e. double stranded circular and covalently closed DNA having more than 1000 base pairs or more than 500 base pairs preferably cannot be part of the nanocarrier system. However, in certain embodiments, it is possible that the entity may also be plasmidic DNA according to the definition given above.
In an embodiment, the entity of the nanocarrier system is a ribonucleic oligonucleotide, i.e. an RNA oligonucleotide. In another embodiment, the ribonucleic oligonucleotide is a messenger RNA (mRNA) or an mRNA analogue, a micro RNA (miRNA), a small interfering RNA (siRNA) or a tiny noncoding RNA (tnRNA).
In another embodiment, the oligonucleotide is a double stranded circular covalently closed deoxyribonucleic or ribonucleic oligonucleotide with a length of ca. 20 to less than 1000 base pairs, preferably of ca. 20 to less than 500 base pairs.
In another embodiment, the ribonucleic oligonucleotide has a length of 8 to 50 bases, preferably 10 to 40 bases, preferably 10 to 30 bases, preferably 12 to 25 bases, preferably 12 to 22 bases.
In any case, the bases which might be part of the nucleotides, nucleosides, or oligonucleotides used as or in the entity of the nanocarrier system, can be natural or non-natural bases. E.g., suited nucleoside analogues include, but are not limited to inosine, nebularine, nitropyrrole, nitroindole, 2-aminopurine, 2,6-diaminopurine, locked nucleic acids (LNAs) nucleosides, peptide nucleic acids (PNAs) nucleosides, purine, hypoxanthine, xanthine, ethanocytosin, 5-methylcytosine, 5-alkynylcytosine, 2,6-diaminopyrimidino, 2,6-diaminopyrazine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine, isoguanine, inosine, 4-acetylcytosine, dihydrouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl), pseudouridine, pseudocytosine, pseudoisocytosine, 5-propynylcytosine, isocytosine, isoguanine, 2-thiopyrimidine, 6-thioguanine, 4-thiothymine, 4-thiouracil, O6-methylguanine, N6-methyladenine, O4-methylthymine, 5,6-dihydrothymine, 5,6-dihydrouracil, 4-methyl-indole, and ethenoadenine.
Accordingly, the nucleotides can be modified in this manner. The oligonucleotides may be LNAs or PNAs. PNA is a polymer of purine and pyrimidine bases which are connected to each other via a 2-amino ethyl bridge. PNA binds sequence specifically with high affinity to an according DNA or RNA complement. In LNA, the 2′-hydroxyl oxygen of ribose is connected to the C-4 atom of the same ribose unit via a methylene bridge. The conformational restriction of LNA compared to RNA or DNA apparently leads to a higher binding affinity.
All possible meanings for the entity as defined above are to be understood as individually disclosed herein and to be optionally combined in any desired manner.
In an embodiment, the nanocarrier system comprises a polyglycerol core in which at least 50%, particularly at least 60%, particularly at least 70%, particularly at least 80%, particularly at least 90%, particularly at least 95%, particularly at least 99%, particularly all of the free hydroxyl groups of the polyglycerol core are substituted by groups of the type X.
In another embodiment, n is preferably 1 to 10, particularly 5.
In another embodiment, the nanocarrier system preferably comprises a compound in which R1 is a methyl residue and R2 is an N-dimethyl ethyl amine residue.
The objective is also attained by a compound having the features of claim 1. Such a compound is suited as entity carrier and has the general formula (I)
with    PG denoting a linear or branched polyglycerol core,    X being
and being covalently bound to a carbon atom of the polyglycerol core, wherein the polyglycerol core carries a plurality of groups of the type X,    R1 being H, linear or branched C1-C10-Alkyl, which may be substituted and/or interrupted by one or more oxygen, sulphur and/or nitrogen atoms, or R3,    R2′ being linear or branched C1-C10-Alkyl, which may be substituted and/or interrupted by one or more oxygen, sulphur and/or nitrogen atoms, or R3,    R3 being
    R4 being H or C1-C4-Alkyl, which may be substituted and/or interrupted by one or more oxygen, sulphur and/or nitrogen atoms, and    n being 1 to 100,wherein R1 and R2′ cannot simultaneously be an ethyl residue.
In an embodiment, the entity to be carried by said compound suited as entity carrier is chosen from the group comprising nucleotides, nucleosides, linear or circular single or double stranded oligonucleotides, oligomeric molecules comprising at least one nucleoside, small pharmacologically active molecules having a molecular mass of not more than 800 g/mol, amino acids, peptides, and metal ions. In an embodiment, the afore-mentioned group does not comprise DNA or plasmidic DNA or essentially completely double stranded nucleic acids or any combination thereof.
E.g., a single nucleoside (of ribonucleic acid or of deoxyribonucleic acid) like uridine or deoxythymidine or a plurality of identical or different nucleosides may serve as entity. Examples of nucleotides are adenosine monophosphate (AMP), adenosine diphosphate (ADP) and adenosine triphosphate (ATP). Also, different metal ions, particularly cations such as Ag+, Ca2+, Cu2+ or Mg2+ ions, or aptamers of peptides or oligonucleotides may serve as entity. In case of the entity being metal ions, preferably a non-covalent, complexed form of interaction between the metal ions and the carrier is established.
Another example of a suited entity are single or double stranded RNA or DNA oligomers of, e.g., 20 to 25 base pairs or bases, respectively, in length. Double stranded RNA is particularly suited. Thus, the compound may also serve as siRNA carrier. Another example of suited entities are chimeric molecules of amino acid residues and nucleosides or nucleotides.
Reference is also made to the explanations given above with respect to the nanocarrier system which are also applicable for the compound and its use.
All possible meanings for the entity as defined above are to be understood as individually disclosed herein and to be optionally combined in any desired manner.
As can be seen from formula (I) and the residue definitions given above, the claimed compound has a polyglycerol (PG) based gene-transfection motif with core-shell architecture. The shells of such motifs can be tailored to contain amines with different numbers of cationic sites for mimicking the activity of polyamines. Since the compounds are based on a PG structure, they provide appreciable clinical compliance.
In contrast to polyamines and other known compounds used as carriers, the novel compounds carry charges at physiological pH only on their surface or shell (namely on nitrogen atoms located on the surface or being part of the shell), whereas the core is comprised of short alkyl chains connected via ether bridges to each other being substantially not charged. The polyglycerol core may be structured in a linear or branched manner. In an embodiment, the structure of the polyglycerol is at least partially branched.
The shell of the polyglycerol-based compounds may have a layered structure due to a repetitive nitrogen-containing motif. E.g., by use of a pentaethylenehexamine residue as shell (as is the case in polyglyceryl pentaethylenehexamine carbamate), a five-fold layered shell is achieved.
The polyglycerol base material can be obtained in a kilogram scale which contains linear monohydroxy and terminal dihydroxy functionalities which can be modified selectively as linkers for diverse organic synthesis.
The polyglycerol core of the claimed compounds is biocompatible. However, by introducing nitrogen-containing shell motifs, the cell toxicity of the compounds is raised. Thus, when designing carriers to be especially used for in vivo applications, care must be taken to keep the cell toxicity of the complete compound at a low level. On the other hand, the transfection efficacy should be as high as possible. Consequently, a balance must be found between toxicity and transfection efficacy. Regarding the claimed compounds, such a balance is established.
Specific, symmetric polyglycerol dendrimers are an example of polyglycerol which can be used for the polyglycerol core of the nanocarrier system or the compound. These dendrimers are highly symmetric. They are generated from smaller molecules by repeated reaction steps, wherein always higher degrees of branching result. At the end of the branches, functional groups are located which are the starting point for further branchings. Thus, with each reaction step, the number of monomeric end groups increases exponentially, leading to a hemicircular tree structure.
In this context, the term “polyglycerol” as used herein includes any substance which contains at least two glycerol units in its molecule and wherein said molecule is characterized by a branched structure. According to the present invention, the term “glycerol unit” does not only relate to glycerol itself but also includes any subunits which are based on glycerol, such as for example:

Preferably, the polyglycerol includes three or more, preferably ten or more, and particularly 15 or more of said glycerol units. The polyglycerol structure can be obtained, e.g., by a perfect dendrimer synthesis, a hyperbranched polymer synthesis or a combination of both and is per se known to a person skilled in the art.
In an alternative embodiment, at least 50%, particularly at least 60%, particularly at least 70%, particularly at least 80%, particularly at least 90%, particularly at least 95%, particularly at least 99%, particularly all of the free hydroxyl groups of the polyglycerol core of the compound are substituted by groups of the type X. The rate of substitution is also referred to as conversion. Thus, if a conversion of 100% is achieved during synthesis, the starting material polyglycerol of the formula PG-(OH)p was reacted to PG-(X)m with m=p. If, e.g., a product of the formula (X)m-PG-(OH)q with m=0.8*n and q=0.2*p is obtained, the conversion is 80%.
As an alternative, n is 1 to 10, particularly 2 to 8, particularly 3 to 6 and in particular 5. As another alternative, R4 is H. If X is
n is 5 and R4 is H, the compound would be polyglyceryl pentaethylenehexamine carbamate.
In another embodiment, R1 is a methyl residue and R2′ is an N-dimethyl ethyl amine residue so that an N,N,N′-trimethylethylenediamine residue is bound to the polyglycerol core structure via one of its nitrogen atoms.
The objective is also achieved by a use of a compound as defined above (with all alternative embodiments) with respect to the claimed compound or of a compound according to general formula (I), wherein X is
according to claim 9. This use is directed to the preparation of a pharmaceutical composition, wherein the compound acts as carrier for an entity, wherein the entity is chosen from the group comprising nucleotides, nucleosides, linear or circular single or double stranded oligonucleotides, oligomeric molecules comprising at least one nucleoside, small pharmacologically active molecules having a molecular mass of not more than 800 g/mol, amino acids, peptides, and metal ions. However, it is mandatory that the entity does not comprise a double stranded circular and covalently closed nucleic acid (preferably DNA or RNA, in particular DNA) having a length of more than 1000 bases or base pairs, preferably of more than 500 bases or base pairs.
Examples of entities are disclosed above. All possible meanings for the entity as defined above are also in the context of the claimed uses to be understood as individually disclosed herein and to be optionally combined in any desired manner.
The pharmaceutical composition may be used to treat diverse diseases, such as diseases which are amenable to treating by gene silencing like, e.g., certain types of cancer. Further, in particular when Cu2+ ions are used as entity, the pharmaceutical composition may serve for slowing down aging.
The objective is also achieved by the use of a compound of the general formula (I) as explained above (with all alternative embodiments) with respect to the claimed compound or of a compound according to general formula (I), wherein X is
as entity carrier for in vitro, in vivo, ex vivo or in situ applications.
In this context, the entity is chosen from the group comprising nucleotides, nucleosides, linear or circular single or double stranded oligonucleotides, oligomeric molecules comprising at least one nucleoside, small pharmacologically active molecules having a molecular mass of not more than 800 g/mol, amino acids, peptides, and metal ions. However, at least in case of R1 and R2 both being an ethyl residue, the entity does not comprise a double stranded circular and covalently closed nucleic acid (preferably DNA or RNA, in particular DNA) having a length of more than 1000 bases or base pairs, preferably of more than 500 bases or base pairs. If the polyglycerol core is substituted by a carbamate residue or a residue of the general structure
plasmidic nucleic acids (i.e. double stranded circular and covalently closed nucleic acids) are also suited as entity in an embodiment.
In an alternative embodiment, the use is directed only to in vitro, ex vivo or in situ applications, but not to in vivo applications. In another alternative embodiment, the use is directed only to in vitro or ex vivo applications, but not to in vivo or in situ applications.
In another embodiment, it is preferred to use the entity carrier for in vivo or in situ applications.
An example of such a use or a use as pharmaceutical composition is to utilize the entity carrier to transport said entity into at least one prokaryotic or eukaryotic cell, in particular into at least one human or animal cell. Transporting said entity in a plurality of entity carriers into a plurality of cells is preferred. Suited animal cells are, e.g., cells of mammals like, e.g., rodents such as rats or mice.
In an alternative embodiment, the entity carrier is used to transport entity into at least one animal cell but not into a human cell. Thus, the use of the compound as entity carrier may be defined as for in vitro, in vivo, ex vivo or in situ applications with respect to animal cells and for in vitro, ex vivo or in situ applications for human cells.
In an embodiment, the use is directed to silence a gene within a cell, preferably a tumor gene, by a ribonucleic acid, such as siRNA.
In another embodiment, the entity carrier bears at least one functional group of the general formula
wherein residues R3 and R4 have the above-defined meanings, and in that said functional group is cleaved from the polyglycerol core of the entity carrier once the entity carrier is located within its target cell. This cleavage results in an even better biocompatibility of the compound, since potentially cytotoxic amine structures of the compound like polyamine or polyethyleneamine structures are separated from the generally biocompatible polyglycerol core structure.
In another embodiment, said cleavage is performed by an enzyme. E.g., the compound may be designed in such a way that an esterase or a carbamate hydrolase may cleave the carbamate bond so that the polyglyceryl core is separated from the surrounding amine-containing surface or shell.
Again, all possible meanings for the entity as defined above are also in the context of the claimed nanocarrier system to be understood as individually disclosed herein and to be optionally combined in any desired manner.
The invention also relates to a kit for performing transfection reactions, comprising a compound of general formula (I) as defined above in any suited formulation. E.g., a formulation as a solution in a phosphate buffered saline (PBS) at pH 7.4 or as a lyophilized product with or without adducts may be suited. Other buffer systems can also be used.
The object is also achieved by a method for silencing a gene in vitro, ex vivo, in situ or in vivo according to claim 15. This method is characterized by applying a nanocarrier system as described above or a compound as described above together with at least one entity to be carried by and bound to the mentioned compound in a covalent, ionic or complexed manner into at least one human or animal cell. Thereby, the entity is a ribonucleic oligonucleotide.
In an alternative embodiment, the method is directed only to in vitro or ex vivo applications, but not to in vivo or in situ applications. In another alternative embodiment, the method is directed only to in vitro, in situ or ex vivo applications, but not to in vivo applications.
The application of the nanocarrier system or the compound/ribonucleic acid is done such that the ribonucleic acid can interact with mRNA being present in said cell.
In another embodiment, it is preferred to use the method for in vivo or in situ applications.
In an embodiment, the gene to be silenced is a tumor related gene.
The object is further achieved by a method for the treatment of cancer according to claim 17. This method is characterized by administering a nanocarrier system as described above or a compound as described above together with at least one entity to be carried by and bound to said compound in a covalent, ionic or complexed manner, to at least one human or animal being. Thereby, the entity is chosen from the group comprising nucleotides, nucleosides, linear or circular single or double stranded oligonucleotides, oligomeric molecules comprising at least one nucleoside, small pharmacologically active molecules having a molecular mass of not more than 800 g/mol, amino acids, peptides, and metal ions
In an embodiment, the treatment of cancer is performed as combination treatment by a combined administering of the nanocarrier system or the compound/entity according to the invention together with a known anti-cancer or an anti-angiogenic drug. Exemplary cytotoxic agents suited as anti-cancer drug include, without limitation, anthracycline antibiotics like doxorubicin and daunorubicin; taxanes like paclitaxel Taxol™, docetaxel; vinca alkaloids like vincristine and vinblastine, anti-metabolites like methotrexate, 5-fluorouracil (5 FU), leucovorin, irinotecan, idarubicin, mitomycin C, oxaliplatin, raltitrexed, tamoxifen and cisplatin, carboplatin, actinomycin D, mitoxantrone or blenoxane or mithramycin.
Examplary anti-angiogenic drugs include, but are not limited to: (1) monoclonal antibodies directed against specific proangiogenic factors and/or their receptors; (avastin, erbitux, vectibix, herceptin) and (2) small molecule tyrosine kinase inhibitors (TKIs) of multiple proangiogenic growth factor receptors (tarceva, nexavar, sutent, iressa). Inhibitors of mTOR (mammalian target of rapamycin) represent a third, smaller category of antiangiogenic therapies with one currently approved agent (torisel). In addition, at least two other approved anti-angiogenic agents may indirectly inhibit angiogenesis through mechanisms that are not completely understood (velcade, thalidomide/celgene). Other anti-angiogenic agents that are suitable for use in the context of embodiments of the invention include, but are not limited to, paclitaxel, 2-methoxyestradiol, prinomastat, batimastat, BAY 12-9566, carboxyamidotriazole, CC-1088, dextromethorphan acetic acid, dimethylxanthenone acetic acid, endostatin, IM-862, marimastat, a matrix metalloproteinase, penicillamine, PTK787/ZK 222584, RPI.4610, squalamine lactate, SU5416, thalidomide, combretastatin, tamoxifen, COL-3, neovastat, BMS-275291, SU6668, anti-VEGF antibody, medi-522 (vitaxin II), CAI, interleukin-12, IM862, amilloride, Angiostatin® protein, angiostatin K1-3, angiostatin K1-5, captopril, DL-alpha-difluoromethylornithine, DL-alpha-difluoromethylornithine HCl, His-Tag® Endostatin™ protein, Endostar™, fumagillin, herbimycin A, 4-Hydroxyphenylretinamide, juglone, laminin, laminin hexapeptide, laminin pentapeptide, lavendustin A, medroxyprogesterone, medroxyprogesterone acetate, minocycline, minocycline HCl, placental ribonuclease inhibitor, suramin, sodium salt suramin, human platelet thrombospondin, neutrophil granulocyte; interferon alpha, beta and gamma; IL-12; matrix metalloproteinases (MMP) inhibitors (e.g. COL3, Marimastat, batimastat); EMD121974 (cilengitide); ZD6474, SU11248, Vitaxin; squalamin; COX-2 inhibitors; PDGFR inhibitors (e.g., gleevec); NM3 and 2-ME2.
Alternative embodiments of the nanocarrier system, the compound or the entity as indicated above are also independently applicable for the claimed methods.
The invention will be explained in the following with reference to figures and examples. This will be done for better understanding of the invention is not intended to limit the scope of protection in any way.
FIG. 1 shows a cut-out of the polyglycerol core of a compound according to general formula (I). The grade of branching of the polyglycerol structure can differ from that depicted in FIG. 1. Also, the molecular mass of a polyglycerol core structure of the claimed invention can be equal to that shown in FIG. 1 or can be lower or higher. During synthesis of the compounds according to general formula (I), substitution reactions take place at free hydroxyl residues (—OH) of the polyglycerol structure. In the abbreviated structure
only a single hydroxyl group is indicated. However, this single hydroxyl group is to be understood as representative of all free hydroxyl groups being present in the polyglycerol.
FIGS. 2 and 3 showing reaction schemes will be explained in conjunction with examples 1 to 3.
FIGS. 4 to 7 showing siRNA silencing efficiencies of two siRNA loaded nanocarriers with respect to different protein expressions will be explained in the context of example 4.
FIGS. 8, 9 and 10A to 10D relating to cytotoxicity experiments will be explained in conjunction with example 5.
FIGS. 11 and 12 relating to an in vivo gene silencing experiment will be explained in conjunction with example 6.
FIG. 13 relating to body weight change of animals after treatment with a nanocarrier will be explained in conjunction with example 7.
FIG. 14 will be explained in conjunction with Example 8.
FIG. 15 will be explained in conjunction with Example 9.
FIG. 16 will be explained in conjunction with Example 10.
FIGS. 17 and 18 will be explained in conjunction with Example 11.